The tear film is necessary for the quality of the visual image and for the protection of the ocular surface, and dry eye states are major causes of ocular morbidity. Deficiencies of the aqueous component of the tear film, which is produced by the lacrimal glands, have been implicated in a large proportion of dry eye cases. Little is known about lacrimal secretory function. Consequently, dry eye treatments must rely on replacement with artificial tears. The working hypothesis for lacrimal fluid secretion to be evaluated in this project is comprised of ion transporters predicted to catalyze the electrolyte flux driving osmotic water secretion in exocrine glands. These include: Na,K-ATPase, sodium, potassium, and chloride conductance pathways, NaCl, NaKCl, and KCl symporters, and parallel Na/H and Cl/anion antiporters. Most conventional methods for studying lacrimal secretion utilize intact tissue or cell preparations, in which it is impossible to measure transport through the apical membranes or to distinguish between alternative mechanisms for coupled sodium and chloride transport in the basal-lateral membranes. Therefore, it has not previously been possible to design definitive tests of the model, to learn how the transport activities are controlled, or to identify specific transport defects causing dry eyes. In the proposed studies, tracer uptake and spectrophotometric methods will be used to characterize transport processes in functional plasma membrane vesicles isolated from the rat exorbital lacrimal gland. This approach will avoid the technical limitations of conventional preparations. The transport methodologies have already been productively applied in vesicle studies of renal and intestinal transport mechanisms. Centrifugation and polymer phase-partitioning methods are available for isolating exorbital gland basal-lateral plasma membranes as sealed vesicles retaining transport systems of the intact cell membrane. The isolation procedures also yield a vesicle population provisionally identified as apical membranes. In parallel with the transport experiments, immunocytochemical studies based on monoclonal antibodies will be performed to verify this population's apical membrane identity.